The Molecular Basis of Temperature Compensation in the Arabidopsis Peter Gould, James C.W. Locke, Camille Larue, Megan Southern, Seth Davis, Shigeru Hanano, Richard Moyle, Raechel Milich, Joanna Putterill, Andrew Millar, et al.

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Peter Gould, James C.W. Locke, Camille Larue, Megan Southern, Seth Davis, et al.. The Molecular Basis of Temperature Compensation in the Arabidopsis Circadian Clock. The Plant cell, American So- ciety of Plant Biologists (ASPB), 2006, 18 (5), pp.1177-1187. ￿10.1105/tpc.105.039990￿. ￿hal-02325735￿

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The Molecular Basis of Temperature Compensation in the Arabidopsis Circadian Clock W

Peter D. Gould,a James C.W. Locke,b,c,d Camille Larue,a Megan M. Southern,c Seth J. Davis,e Shigeru Hanano,e Richard Moyle,f,g Raechel Milich,f Joanna Putterill,f Andrew J. Millar,d,h,1 and Anthony Halla,1 a School of Biological Sciences, University of Liverpool, Liverpool L69 7ZB, United Kingdom b Department of Physics, University of Warwick, Coventry CV4 7AL, United Kingdom c Department of Biological Sciences, University of Warwick, Coventry CV4 7AL, United Kingdom d Interdisciplinary Programme for Cellular Regulation, University of Warwick, Coventry CV4 7AL, United Kingdom e Max Planck Institute for Plant Breeding, Cologne D-50829, Germany f University of Auckland, School of Biological Sciences, Private Bag 92019, Auckland, New Zealand g Botany Department, School of Integrative Biology, University of Queensland, Brisbane Qld 4072, Australia h Institute of Molecular Plant Sciences, University of Edinburgh, Edinburgh EH9 3JH, United Kingdom

Circadian clocks maintain robust and accurate timing over a broad range of physiological temperatures, a characteristic termed temperature compensation. In , ambient temperature affects the rhythmic accumulation of transcripts encoding the clock components TIMING OF CAB EXPRESSION1 (TOC1), GIGANTEA (GI), and the partially redundant CIRCADIAN CLOCK ASSOCIATED1 (CCA1) and LATE ELONGATED HYPOCOTYL (LHY). The amplitude and peak levels increase for TOC1 and GI RNA rhythms as the temperature increases (from 17 to 278C), whereas they decrease for LHY. However, as temperatures decrease (from 17 to 128C), CCA1 and LHY RNA rhythms increase in amplitude and peak expression level. At 278C, a dynamic balance between GI and LHY allows temperature compensation in wild-type plants, but circadian function is impaired in lhy and gi mutant plants. However, at 128C, CCA1 has more effect on the buffering mechanism than LHY, as the cca1 and gi mutations impair circadian rhythms more than lhy at the lower temperature. At 178C, GI is apparently dispensable for free-running circadian rhythms, although partial GI function can affect circadian period. Numerical simulations using the interlocking-loop model show that balancing LHY/CCA1 function against GI and other evening-expressed genes can largely account for temperature compensation in wild-type plants and the temperature- specific phenotypes of gi mutants.

INTRODUCTION clock in other organisms. The loop comprises two myb tran- scription factors, LATE ELONGATED HYPOCOTYL (LHY) and The circadian clock is an endogenous 24-h timer found in most CIRCADIAN CLOCK ASSOCIATED1 (CCA1), which are pro- eukaryotes and in photosynthetic bacteria. The clock drives posed to function by repressing the expression of TIMING OF rhythms in the physiology, biochemistry, and metabolism of the CAB EXPRESSION1 (TOC1), which encodes a pseudoresponse organisms (reviewed in Hall and McWatters, 2006). Circadian regulator of unknown biological function. TOC1, in turn, activates rhythms are defined as rhythms that persist in constant condi- LHY/CCA1 (Alabadi et al., 2001). It is clear that this model is not tions with a periodicity of ;24 h, that can be entrained to local sufficient to explain current experimental data, as abnormal time, and that are capable of maintaining a constant periodicity rhythms persist in plants lacking both LHY and CCA1 or in those over a broad range of physiological temperatures. Intriguingly, lacking TOC1 (Alabadi et al., 2002; Mizoguchi et al., 2002; Locke although these characteristics of the clock are shared across et al., 2005). Mathematical simulations incorporating current taxonomic groups, there is little conservation of the molecular experimental data have led to the proposal of a model compris- components used (reviewed in Harmer et al., 2001). ing two interlocking feedback loops, with GIGANTEA (GI) and In Arabidopsis thaliana, a transcription feedback loop model TOC1 as candidates for components of a second loop (Locke has been proposed for the clock, similar to the architecture of the et al., 2005). One of the key characteristics of all circadian rhythms is that 1 To whom correspondence should be addressed. E-mail andrew. they are able to maintain robust rhythms with a period close to [email protected] or [email protected]; fax 44-0131-6505392 24 h over a broad range of physiological temperatures; this property or 44-0151-7954403. The authors responsible for distribution of materials integral to the is termed temperature compensation. In Drosophila, studies iden- findings presented in this article in accordance with the policy described tified a role for the key clock component PERIOD (PER) in tem- in the Instructions for Authors (www.plantcell.org) are: Andrew J. Millar perature responses, including temperature-dependent variation ([email protected]) and Anthony Hall (anthony.hall@liverpool. in Per properties (Sawyer et al., 1997) and differential ac.uk). W Online version contains Web-only data. splicing of the Per RNA (Majercak et al., 1999). In Neurospora, Article, publication date, and citation information can be found at two isoforms of the central clock component Frequency (FRQ) www.plantcell.org/cgi/doi/10.1105/tpc.105.039990. have been implicated in maintaining rhythm robustness over a 1178 The Plant Cell

physiological range of temperatures: mutants lacking either form addition to this role in temperature compensation, GI also plays a are rhythmic in only a limited temperature range (Liu et al., 1997). critical role in extending the temperature range over which robust Both studies suggest the hypothesis that temperature compen- and accurate rhythmicity can be maintained. sation is an intrinsic feature of the central clock components, rather than a result of additional regulators. RESULTS To identify components of the temperature compensation mech- anism in the model plant Arabidopsis, a quantitative genetic GI Plays a Critical Role in Maintaining Rhythmicity in approach has been used. This exploits accession-specific var- Leaf Movement at Higher Temperatures iations in the pattern of temperature compensation for rhythms of leaf movement between Columbia (Col) and Landsberg erecta The QTL PerCv1b was identified as a locus involved in temper- (Ler) and between Ler and Cape Verde Islands (Cvi). This strategy ature compensation of the circadian clock from a screen of Cvi 3 uncovered a number of quantitative trait loci (QTL) across the Ler recombinant inbred lines. Further mapping of this QTL using genome. The genes underlying some of these, such as the flow- near isogenic lines localized the QTL to an ;900-kb region of ering regulator FLOWERING LOCUS C (Edwards et al., 2006), 1. Within this region was the flowering-time had no known function in the Arabidopsis clock mechanism. By GI. Sequencing GI from Ler and Cvi revealed a number of poly- contrast, further characterization of one of these QTL, PerCv1b, morphisms, two of which resulted in amino acid substitutions identified GI as a candidate for the PerCv1b trait (Edwards et al., (Edwards et al., 2005). This, together with the period effects of 2005). the gi mutant (Park et al., 1999), confirmed GI as a strong can- The gi alleles were originally identified as supervital mutants didate for a gene involved in the temperature compensation having increased fitness as measured by high fecundity, result- mechanism. ing in increased seed yield per plant. The basis for this was the To identify a role for GI in the temperature buffering of the large rosettes and late flowering of the mutants (Re´ dei, 1962). circadian clock, circadian rhythms of leaf movement in the gi null More recently, GI has been identified as acting upstream of mutant gi-11 over a range of temperatures (12, 17, 22, and 278C) CONSTANS in the photoperiodic regulation of flowering (Suarez- were characterized. The gi-11 null mutant is a T-DNA insertion Lopez et al., 2001). The GI gene encodes a 127-kD nuclear pro- mutation and has been described previously (Richardson et al., tein of 1173 amino acids (Fowler et al., 1999; Park et al., 1999; 1998; Fowler et al., 1999); furthermore, the late-flowering phe- Huq et al., 2000). GI is conserved in plants; however, GI homo- notype of the gi-11 mutation could be complemented with a wild- logs appear to be absent from the genomes of the microalga type GI cDNA (see Supplemental Figure 1 online). Both gi-11 and Chlamydomonas (Mittag et al., 2005) and those of human, wild-type plants were grown under 12-h-light/12-h-dark cycles mouse, and Drosophila. GI is expressed throughout the plant (12L:12D) at 228C to entrain the clock and were then transferred and is regulated by the circadian clock, with a maximal level of to constant light at 12, 17, 22, or 278C. The free-running periods expression 12 h after dawn (Fowler et al., 1999). Mutations in the of the leaf movement rhythms were measured. Figure 1A shows clock-affecting genes LHY, CCA1, and TIME FOR COFFEE alter that at 178C, the gi mutant (open triangles) and the wild type GI RNA expression levels (Mizoguchi et al., 2002; Hall et al., (closed squares) had no significant period difference. However, 2003). In addition to its role in flowering, GI has also been as the temperature increased, the period of gi became signifi- phenotypically characterized as affecting the circadian clock, as cantly shorter than that of the wild type. A significant period the gi-1 mutant causes a short-period phenotype for both leaf shortening was also observed in response to a decrease in movement and CAB: (LUC) expression. Another temperature. This temperature-dependent effect on the free- allele, gi-2, causes a similar short-period phenotype for leaf move- running period is consistent with GI playing a function in the tem- ment, although CAB:LUC expression oscillates with a long perature compensation mechanism of the Arabidopsis clock. period (Park et al., 1999). The gi mutants cause a reduction in In addition to a shortening of the period, a temperature- the expression of CCA1 and LHY (Fowler et al., 1999). Recent dependent increase in the variance of period was seen for the investigations (Mizoguchi et al., 2005), together with this study, gi mutant, with the variance increasing from 3.1 at 178C to 18.0 at suggest that the flowering and circadian functions of GI are 278C(F test, P ¼ 1.8 3 109). Although an increase in the variance distinct. of period with temperature was also observed for the wild type, In this study, we demonstrate that the gene GI plays a critical this was not of the same magnitude (from 1.9 at 178C to 3.8 at role in increasing the temperature range permissive for rhyth- 278C; F test, P ¼ 4.1 3 103). The huge increase in the variance of micity, extending the temperature range over which both the period in the gi mutant at 278C demonstrates a breakdown in the robust rhythms of CAB expression and leaf movement rhythms precision of the clock. This reduced precision could clearly be can be maintained. We go on to investigate, first, how key com- seen when period estimates for each leaf movement rhythm were ponents of the wild-type circadian mechanism (GI, LHY, CCA1, plotted against the relative amplitude error (Figure 1B), a mea- and TOC1) change in response to altered temperature. Second, sure of rhythm robustness varying from 0 (a perfect fit to the we describe how this temperature-specific regulation is altered cosine wave) to 1 (not statistically significant). For wild-type in the gi mutant, identifying a mechanism for the temperature- period estimates, the associated relative amplitude errors re- specific phenotype of GI. We conclude that a dynamic balance mained low, with all 60 points clustering tightly at each temper- between LHY and GI is key for the effective temperature com- ature (Figure 1B). However, for the gi mutant, as temperature pensation of the circadian clock at high temperatures, whereas increased, both the relative amplitude error and the range of at low temperature LHY appears to be substituted by CCA1.In period estimates increased. Similarly, as temperature decreased Temperature Compensation of the Clock 1179

to <178C, the variability and relative amplitude errors increased (Figure 1B). Similar results were seen for the gi-3 allele in the Ler background, with temperature-dependent effects on both period and rhythm robustness observed (see Supplemental Figure 2 online). However, unlike the null mutant, the period of gi-3 was not wild type at 178C. The gi-3 mutation introduces a stop codon at the 39 end of the gene; therefore, a truncated GI protein can be produced. Furthermore, the expression of GI mRNA in the gi-3 mutant had only been reduced by half (Fowler et al., 1999). Similarly, another gi allele isolated in our laboratory, gi-611, which lacked the characteristic flowering phenotype of the standard gi mutants, also had a temperature-dependent period phenotype, although it did not have a reduced robustness phenotype (see Supplemental Figure 3 online). Together, the leaf movement anal- ysis suggests a role for GI not only in buffering the period of the clock against temperature change but also in extending the range of temperature over which rhythmicity can be maintained.

GI Plays a Comparable Role Maintaining the Rhythmicity of Expression of CAB:LUC at High and Low Temperatures

It is clear that GI functions to extend the temperature range at which accurate leaf movement rhythms can be maintained. To investigate whether this extended to other rhythms, the CAB: LUC marker was used to assay the rhythmic expression of CAB in the gi-11 background in plants entrained at 228C and then free run in constant light at 12, 17, or 278C. After transfer to 178C, the rhythmic expression patterns of CAB in the wild type and gi-11 were almost identical (Figure 2A), with no significant differ- ence in period and >98% of gi-11 seedlings producing periods within the circadian range (15 to 35 h). The only difference in the circadian phenotype between gi-11 and the wild type at 178C was the low amplitude of CAB expression in gi-11 and an increase in the variance of period (period variance of 0.16 for the wild type and 1.14 for gi; F test, P ¼ 3 3 1012). Thus, at 178C, the circadian clock is able to sustain rhythms of CAB expression even in the absence of GI. However, upon transfer of gi-11 seedlings from 22 to 278C, CAB expression rapidly dampened, and by 48 h expression was arrhythmic (Figure 2A). This was Figure 1. Temperature Compensation of Leaf Movement Rhythms for reflected by the fact that only 30% (8 of 26) of gi-11 seedlings Wassilewskija (Ws) Wild Type and Ws gi-11. produced periods for CAB expression within the circadian range, Seedlings were entrained under 12L:12D cycles for 7 d, after which they all with relative amplitude errors of >0.5, indicative of weak were transferred to constant light at 12, 17, 22, or 278C, at which rhythms rhythms. By contrast, 100% (31 of 31) of wild-type seedlings of leaf movement were assayed. For each temperature, 25 to 30 wild- were scored as producing robust rhythms in CAB expression at type plants and 25 to 30 gi mutant plants were assayed, corresponding 278C. This response was mirrored when gi-11 plants were to 50 to 60 leaf movement rhythms. transferred from 22 to 128C, with CAB expression rapidly be- (A) Variable-weighted mean of period estimates plotted against temper- ature. Closed squares, wild type; open triangles, gi-11. Error bars coming arrhythmic (Figure 2A) and only 35% (15 of 43) of represent variance-weighted SE. Asterisks indicate t test P values: seedlings producing rhythms within the circadian range. These * P < 0.05, ** P < 0.01. observations are consistent with the leaf movement analysis in (B) Period estimates for individual leaves plotted against their relative that GI functions to extend the temperature range permissive for amplitude errors (Rel. Amp. Error). Closed squares, wild type (n ¼ 50 to rhythmicity. Furthermore, its similar effect on these two outputs 60); open triangles, gi-11 (n ¼ 50 to 60). This experiment was performed is consistent with GI acting on the central clock. independently three times at each of the four temperatures; the results shown are representative. Temperature Regulation of Clock-Related

To investigate how key molecular clock components respond to ambient temperature in wild-type plants, the accumulation of transcripts that encoded CCA1, LHY, and TOC1 was measured. 1180 The Plant Cell

Figure 2. GI Is Required for the Rhythmicity of CAB at 12 and 278C. Transgenic seedlings carrying the CAB:LUC reporter gene were entrained under 12L:12D cycles for 7 d, after which the seedlings were transferred to 12, 17, or 278C and constant red and blue light. Luminescence was monitored in the wild-type Ws (closed squares) and gi-11 (open triangles). For both the wild type and gi-11, expression was monitored in at least three independently transformed lines. (A) Plots represent the normalized average expression of 8 to 27 single seedlings of one representative transformed line. Error bars indicate SE. (B) Summary of the mathematical analysis of the expression rhythms of the seedlings, represented by plots of period estimates plotted against the respective relative amplitude errors (Rel. Amp. Error) for wild-type Ws and gi-11.At278C, wild-type n ¼ 31, gi-11 n ¼ 26; at 178C, wild-type n ¼ 116, gi-11 n ¼ 51; at 128C, wild-type n ¼ 52, gi-11 n ¼ 43.

Plants were entrained for 7 d with 12L:12D cycles before transfer (Figure 4). The reciprocal was observed for LHY levels, with the to 12, 17, or 278C and constant light. Transcript abundance was amplitude of the oscillation of LHY decreasing at 278C. CCA1 then assayed, starting at 72 h after transfer, every 4 h over a 24-h expression showed no significant change with increased tem- period. Figures 3A to 3C compare transcript abundance and perature. The difference in response of LHY and CCA1 was con- expression patterns of plants free-running at 17 and 278C. At sistent with the two genes not playing completely redundant 278C, the amplitude of the TOC1 expression rhythm was almost roles. A temperature-dependent increase in clock components, double that at 178C, with the trough level of expression remaining similar to that seen for TOC1, has also been observed in Neu- constant but the peak level increased. Also, the shoulder seen rospora, in which the amount/amplitude of FRQ protein increased after the peak of expression was lost at 278C. A temperature- with temperature (Liu et al., 1998). A comparison of the free- dependent increase in expression was also observed for GI running clock at 17 and 128C revealed no significant differences Figure 3. The Clock Components TOC1, LHY,andCCA1 Have Altered Amplitudes in Response to Temperature and GI Function.

Seedlings were grown at 228C in 12L:12D conditions for 7 d; the seedlings were then transferred to constant light and 12, 17, or 278C. They were harvested after 72 h and every 4 h thereafter for the next 24 h. Total RNA was assayed by real-time quantitative PCR, and the accumulation of TOC1 ([A] and [D]), LHY ([B] and [E]), and CCA1 ([C] and [F]) was measured relative to an internal UBIQUITIN (UBQ) control. The plots compare the accumulation of mRNAs at 278C (open symbols) with that at 178C (closed symbols) ([A] to [C])orat178C (closed symbols) with that at 128C (open symbols) ([D] to [F]). Expression in the gi-11 background is represented by triangles, and that in the wild type is represented by squares. Error bars indicate SE. Each point represents the average of three biological repeats, with each biological repeat made up of three technical repeats. 1182 The Plant Cell

temperature-dependent, we compared the expression profiles of TOC1, CCA1, and LHY in wild-type and gi-11 seedlings free- running in constant light at 27 and 178C, as described above (Figure 3). At 178C, TOC1 expression levels were identical in both the gi-11 and wild-type plants. At 278C, TOC1 levels failed to increase in the gi-11 background and were expressed at an almost constant level. At 178C, the expression of LHY was lower in the gi mutant, and this may contribute to the reduction in amplitude of the CAB:LUC oscillations observed in gi-11 at 178C. At 278C, the expression level and amplitude of LHY were further significantly reduced. For CCA1 expression, in contrast with LHY, the level, amplitude, and expression profile were identical in the gi mutant and the wild type at 178C, demonstrating a clear difference in the regulation of the LHY and CCA1 genes by GI. At 278C in the wild type, CCA1 expression was temperature- independent, whereas in the gi mutant, expression became Figure 4. GI mRNA Expression Is Weakly Temperature-Regulated. temperature-dependent, with levels and amplitude of CCA1 decreasing at 278C. Together, our expression analysis of key Wild-type seedlings were grown at 228C in 12L:12D conditions for 7 d, and the seedlings were then transferred to constant light and 128C (open clock components suggests a mechanism whereby GI main- squares), 178C (closed squares), or 278C (closed triangles). They were tains rhythmicity and accuracy at higher temperatures by the harvested after 72 h and every 4 h thereafter for the next 24 h. Total RNA temperature-dependent regulation of TOC1, thereby maintaining was assayed by real-time quantitative PCR, and the accumulation of GI sufficient levels/amplitude of CCA1 and LHY to sustain accurate was measured relative to an internal UBQ control. Error bars indicate SE. and robust clock function. Each point represents the average of three biological repeats, with each We also tested changes in gene expression in CCA1, LHY,and biological repeat made up of three technical repeats. TOC1 in the wild type and the gi mutant at 128C (Figure 3). Little change in the expression of clock components in response to in the expression of TOC1. However, although less pronounced decreases in temperature was observed in the wild type; however, than the effect of high temperature, peak levels of CCA1 and LHY in the gi mutant, the expression of both CCA1 and TOC1 de- did increase at 128C compared with 178C, and the peak level of creased, with the expression of TOC1 becoming biphasic, with two GI decreased (Figure 4). Similarly, a reciprocal decrease in GI peaks of expression only 12 h apart. This was not observed for was also seen (Figures 3D to 3F). CCA1 or LHY, consistent with a breakdown of the feedback Although only subtle changes in the regulation of clock com- regulatory loop composed of CCA1/LHY and TOC1 described by ponents were observed between 17 and 128C, in a microarray Alabadi et al. (2001). The expression of LHY remained at a similar experiment comparing seedlings grown at 22 and 48C, tran- level at both17 and 128C, although both CCA1 and LHY expression scripts encoding many of the known clock components rapidly had an altered phase, suggesting that the period length of CCA1 dampened to a high level upon transfer to 48C (NASCArrays and LHY is shorter at 128C, which is consistent with the short- experiment reference number NASCARRAYS-138; see Supple- period phenotype of rhythms of leaf movement (Figure 1A). mental Figure 4 online). Although this microarray analysis is consistent with a loss of clock function at 48C, a longer time course will be required to confirm this finding. However, this The Interlocking Loop Model Supports GI Function in apparent loss of rhythmicity has also been observed in chestnut Temperature Compensation (Castanea sativa), in which the levels of Cs LHY and Cs TOC1 dampened to a high level upon transfer to 48C, with LHY failing to GI is proposed to contribute to a key component, Y, of the inter- repress TOC1 levels (Ramos et al., 2005). locking loop model for the Arabidopsis clock mechanism (Locke et al., 2005). Therefore, we tested whether the observed temperature-dependent effects of GI were consistent with the Temperature-Dependent GI Function at the Molecular Level function of Y in this model. Without explicit modeling, it would be difficult to determine whether this was the case or whether our Both our leaf movement experiments and CAB:LUC expression experimental data reflected additional temperature-dependent data suggest that GI functions to maintain accurate and robust functions of GI. mRNA expression profiles for TOC1 and LHY/ rhythmicity at high and low temperatures. Moreover, the effect CCA1 at 27, 17, and 128C for both wild-type and gi genotypes on multiple outputs would support the hypothesis that GI acts were simulated (see Methods; see also Supplemental Methods directly on the central clock at higher and lower temperatures. and Supplemental Figures 5 and 6 online). As temperature Previous studies investigating GI’s role in the clock found that effects were not explicitly included in the model, a qualitative fit CCA1 and LHY levels are reduced in plants grown under long was obtained by varying only the transcription rates of the model days at 228C in the gi-3 mutant. This finding supports a function components LHY and Y. LHY in the model represents both LHY for GI in the regulation of CCA1 and LHY (Fowler et al., 1999). To and CCA1. Y represents GI, although unidentified genes with determine whether GI’s effects on the key clock components are functions that overlap with GI may also contribute to Y. Temperature Compensation of the Clock 1183

The wild type at 278C was modeled as having reduced LHY DISCUSSION expression (Figure 3), which would shorten the circadian period if this were the only effect of temperature. Higher GI expression in Our characterization of key components of the circadian clock the model (following the trend in Figure 4) compensated for the over a range of temperatures has uncovered temperature- reduced LHY levels, substantially restoring the period length and dependent changes in their gene expression. We found that lev- also recapitulating several features of the molecular data in els of LHY mRNA decrease with temperature increases and that Figure 3 (see Supplemental Methods and Supplemental Figure 5 this was counterbalanced by increases in TOC1 and GI. By con- online). In particular, a gi mutation in the model had a more severe trast, CCA1 levels changed little between 27 and 178C; however, low-amplitude phenotype at 278C compared with the standard we did see an increase at lower temperatures (Figures 3 and 4). temperature (see Supplemental Figure 5 online), consistent with This balance between clock components is consistent with the the experimental data (Figures 1 and 2). The wild type at 128C antagonistic balance model of temperature compensation put was modeled by increasing the transcription rate of LHY,asLHY forward by Ruoff et al. (1997). We tested whether these new data RNA levels are increased at lower temperatures (see Supple- could fit within the constraints of a proposed interlocking-loop mental Figure 3C online). GI RNA levels were little reduced at model of the circadian clock, incorporating GI (Locke et al., 2005). 128C (Figure 4), but reducing Y transcription rates in the model First, we considered compensation of the clock in response to again compensated the circadian period. The simulated gi mu- increasing temperature. The effects of increases in temperature tation again recapitulated the observed effects of the gi mutant at could be simulated by reducing LHY transcription rates, causing 128C (see Supplemental Figure 6 online). We propose that the a shortening of the period; however, this could be balanced by reduced Y function that was required to simulate the wild type at increasing GI transcription levels. Although the experimental 128C might represent downregulation of another gene that con- increases in GI expression were not pronounced, there was a tributes to Y, overlapping with GI function (see Supplemental gradual increase in peak expression with temperature (Figure 4). Methods online). Altering only the LHY and Y transcription rates It has been shown that the GI protein oscillates and that this is was sufficient to recreate the major effects of temperature on regulated at the posttranscriptional level (David et al., 2006). clock gene expression, showing that the temperature-dependent Therefore, it is possible that levels of GI could also be modulated effects of GI proposed here are consistent with GI’s location as a by temperature. This is consistent with the temperature com- component of Y in the two-feedback-loop model. pensation mechanisms of other organisms (Rutila et al., 1996; Liu et al., 1997; Lahiri et al., 2005). By altering the transcription parameters of GI and LHY alone, the period of the model clock LHY and CCA1 Contribute Differentially to system was balanced and there was little difference in the phase Temperature Compensation of either LHY or TOC1 mRNA with temperature (see Supplemen- Our data suggest that a dynamic balance between LHY and GI tal Figure 5 online). Together, the simulation and experimental functions maintains robust and accurate rhythmicity at higher data are supportive of the conclusion that LHY and GI form a temperatures and that LHY function is distinct from CCA1 in this dynamic counterbalance to temperature-compensate the clock respect. To confirm the function of LHY in this mechanism, we at high temperature. tested temperature compensation in the lhy and cca1 loss-of- In support of this dynamic counterbalance hypothesis, we function mutants. Both lhy and cca1 maintained robust rhythms show that a null gi mutant (gi-11) has a temperature-dependent of CAB:LUC expression at 17 and 278C (Figure 5). Both mutants circadian phenotype, affecting both robustness and period (Fig- had the same short-period phenotype at 178C (wild type, 23.9 h; ures 1 and 2). In gi-11, the counterbalance between GI and LHY is cca1, 22.8 h; lhy, 22.8 h). At 278C, a period difference of ;1 h was lost and LHY RNA levels decrease at 278C. A similar temperature maintained between the wild type (23.0 h) and cca1 (22.2 h), compensation phenotype was also observed in the gi-611 and whereas in the lhy mutant, the period difference increased to gi-3 alleles; however, the partial gi-611 mutation does not display nearly 2 h (wild type, 23.0 h; lhy, 21.1 h) (Figure 5). The lhy mutant, the loss of rhythm robustness with temperature (see Supple- therefore, has a shorter period than cca1 (Student’s t test, P < mental Figure 3 online). The lhy loss-of-function mutant likewise 0.01) at 278C, supporting a role for LHY in the temperature shows a strong period alteration at high temperature (Figure 5). compensation mechanism. This was suggested previously by This proposal can account for temperature compensation at analysis of natural genetic variation (Edwards et al., 2005), as 278C, but our experimental data show that different processes LHY was noted as a candidate gene for the temperature-specific are involved at low temperature. Specifically, between 17 and QTL PerCv1a and PerCola.At128C, the period of the wild type 128C, GI expression is little altered in the wild type, and the (23.3 h) differed from that of lhy (21.6 h) by 1.7 h. In cca1, gi mutation reduces the expression of CCA1 rather than LHY. however, the rhythm of CAB:LUC expression was dampened Furthermore, at 128C, the cca1 loss-of-function mutant alters (Figure 5), and the period difference between the wild type period more severely than the lhy mutation and also reduces (23.3 h) and cca1 (20.0 h) was 3.3 h. This temperature-dependent rhythmic robustness, the reverse of the situation at 278C. At shortening of the circadian period and the reduction in rhythm low temperature, our data are consistent with CCA1 replacing robustness almost phenocopied the effect of gi-11 at 128C LHY in temperature compensation of the clock. The switch in (Figures 1 and 2). At lower temperatures, our data indicate that the apparent importance of these two transcription factors CCA1 plays a greater role than LHY in temperature compensa- may reflect unknown differences in temperature-dependent tion and the maintenance of rhythm robustness, confirming that biochemical properties of the two . GI participates by CCA1 and LHY functions are not completely overlapping. regulating the relative expression levels of CCA1 and LHY in a 1184 The Plant Cell

Figure 5. The lhy Mutant Is Compromised in Its Ability to Buffer the Clock against Increases in Temperature, and the cca1 Mutant Is Compromised in Its Buffering Capacity at Lower Temperatures. Transgenic seedlings carrying the CAB:LUC reporter gene were entrained under 12L:12D cycles for 7 d, after which the seedlings were transferred to 12, 17, or 278C and constant red and blue light. Luminescence was monitored over the subsequent 96 h. (A) Plots represent average normalized expression of CAB in wild-type Ws (closed squares), the cca1 mutant (open triangles), and the lhy mutant (open circles) at 12, 17, or 278C. Mutant and wild-type expression was monitored in at least four independently transformed lines, and the plots show average expression in representative CAB:LUC lines. (B) Summary of the mathematical analysis of the expression rhythms of the seedlings, represented by plots of period estimates plotted against the respective relative amplitude errors for wild-type Ws (closed squares), cca1 (open triangles), and lhy (open circles). Temperature Compensation of the Clock 1185

temperature-dependent manner, as demonstrated by the effects might be predicted from their short-period phenotype (Yanovsky of the gi mutation, thus contributing to the stable period over a and Kay, 2002). A similar conclusion has been drawn by Mizoguchi wide range of temperatures. The qualitative effect of decreases et al. (2005) based on the characterization of a GI overexpressor in temperature could be simulated by increasing LHY transcrip- and gi-3. They demonstrated a disproportionate effect of both GI tion rates, causing an increase in the period, which could be overexpression and the gi-3 mutation on the control of circadian balanced by decreasing the transcription rates of components of clock–regulated flowering genes compared with other circadian Y redundant to GI (see Supplemental Methods Figure 6 online). clock–regulated genes. In addition to its effect on period, the gi null mutant affects Here, we have shown that GI plays a critical role in temperature the robustness of circadian rhythms, an effect not shared by the compensation of the clock and in extending the range of tem- partial gi-611 allele. The lhy mutant at 278C affects only the peratures at which rhythmicity can be maintained. We recently circadian period, not rhythmic robustness, but cca1 at 128Caffects demonstrated that having a robust and accurate clock increases both, phenocopying the gi mutant. This difference between the photosynthesis and productivity in Arabidopsis (Dodd et al., gi and lhy mutants suggests that the effect of GI on rhythm 2005); thus, our new insight into how the clock maintains ro- robustness at high temperature functions via a mechanism that is bustness and accuracy at high temperatures is likely to have less dependent on LHY than the effect on circadian period. implications for enhancing the performance of plants at higher Simulating the effects of temperature by altering LHY and Y and lower temperatures. It is feasible that this could be used to expression showed that these counterbalancing effects were extend the geographical range of crops and potentially allow the largely sufficient to replicate the observed effects of temperature development of new varieties able to cope better with global on clock gene expression in the wild type. The simulations also climate change. confirmed that the effects of the gi mutation on the clock at all temperatures are consistent with the identification of GI as part of Y. Temperature, in fact, will affect many processes in addition to METHODS LHY and Y transcription rates (potentially, all 61 kinetic param- eters in the model). Obtaining simulations that exactly fit the data Plant Material would likely require all of the parameters to be estimated afresh Arabidopsis thaliana gi-11 was isolated in a screen of T-DNA insertion for each temperature. To constrain the estimation, most of the lines described by Richardson et al. (1998) and Fowler et al. (1999). The experimental results that were used in developing the original CAB:LUCþ transgene, in the Ws background, was as described by Hall model would also have to be replicated at 12 and 278C. The et al. (2002). The cca1-11 and lhy-21 mutants were isolated from the current clock model must also be extended to account for our Arabidopsis Functional Genomics Consortium population (Krysan et al., temperature-specific data. For example, LHY and CCA1 were 1999). Single mutants have been described (Hall et al., 2003). The modeled as a single gene and must be separated to explore the mutants gi-611 and gi-596 were isolated in a screen for mutants with differences in their functions at high and low temperature. altered temporal expression of CAB from an ethyl methanesulfonate population of the Ws CAB:LUC transgenic line 6A. This role of GI in extending the temperature range over which robust rhythms can be maintained is analogous to that of the two alternatively spliced forms of the Neurospora clock gene FRQ, Growth Conditions 1-989 100-989 FRQ and FRQ (Garceau et al., 1997). Both the long For luminescence, RNA extraction, and leaf movement analysis, seed- and short forms of FRQ are capable of restoring rhythmicity in a lings were first surface-sterilized in 70% ethanol for 2 min, immediately frq null strain; however, the long form is unable to sustain rhyth- followed by 50% bleach for 10 min. They were then rinsed twice in sterile micity at low temperatures, and the short form is unable to distilled water and resuspended in 0.15% agar. The seedlings were then sustain rhythmicity at high temperatures (Liu et al., 1997). Thus, sown on Murashige and Skoog medium containing 3% sucrose and 1.5% the two spliced forms together extend the range of temperatures agar. Seeds were kept at 48C for 2 d and then grown in 12L:12D cycles of 2 1 over which rhythms can be maintained. From our data, it is not 80 mmolm s in a Sanyo MLR350 plant growth chamber. Tempera- tures both during entrainment and during experiments were logged using clear how GI extends the range of rhythmicity. However, it is Hobo temperature loggers (Onset Computer). possible that the GI/TOC1 loop of the interlocking-loop model proposed by Locke et al. (2005) plays an important function in stabilizing the clock at higher temperatures. Rhythm Analysis Previously, it was demonstrated that the flowering phenotype Leaf movement rhythms were measured using a time-lapse video imag- of gi mutants is temperature-independent, with the exception of ing system as described by Edwards et al. (2005). However, instead of gi-2 (Araki and Komeda, 1993). Here, we demonstrate that the Ultra-track cameras, Sony Exwave HAD cameras (Sovereign Interna- circadian phenotype of gi alleles is temperature-dependent. tional) were used, having high resolution and allowing the number of Together, these two observations suggest that the circadian and plants assayed on one plate to be increased from 12 to 15. Luminescence flowering phenotypes of gi mutants result from distinct functions levels were analyzed using an ORCA-II-BT 1024 16-bit camera cooled to 808C (Hamamatsu Photonics). The camera was housed on top of a of GI. This conclusion is further supported by the isolation of two Sanyo MIR-553 cooled incubator maintaining a uniform temperature new gi alleles, gi-611 and gi-596 (see Supplemental Figure 60.58C (Sanyo Gallenkamp). Illumination was provided by four red/blue 1 online). These have short- and long-period circadian pheno- light-emitting diode arrays (MD Electronics). Image acquisition and light types, respectively, but neither shows the striking late-flowering control were driven by WASABI imaging software (Hamamatsu Photon- phenotype of other gi alleles under long photoperiods. Indeed, ics). The images were processed using Metamorph 6.0 image-analysis gi-611 mutants flowered early in short photoperiods, which software (Molecular Devices). Individual period estimates were generated 1186 The Plant Cell

by importing data into BRASS (available from www.amillar.org) and using Supplemental Figure 3. Amino Acid Substitutions in the N-Terminal BRASS to run fast Fourier transform nonlinear least-squares analysis Half of the GI Protein Can Lengthen or Shorten Circadian Period but programs (Plautz et al., 1997) on each data trace to generate period Do Not Result in Late Flowering. estimates and relative amplitude errors. Supplemental Figure 4. At 48C, the Oscillations of Key Clock Genes Dampen Rapidly. RNA Analysis Supplemental Figure 5. Modeling the Temperature Compensation Plants entrained in 12L:12D for 7 d at 228C were transferred to constant Mechanism at High Temperatures. light at 12, 17, 22, and 278C. After 72 h of free-running conditions, ;100 Supplemental Figure 6. Modeling the Temperature Compensation seedlings were harvested every 4 h for 24 h and frozen in liquid nitrogen. Mechanism at Low Temperatures. Total RNA was extracted and treated with DNase using RNeasy plant mini Supplemental Methods. kits (Qiagen) according to the manufacturer’s instructions. A 2-mg portion was reverse-transcribed using the Advantage-for-PCR kit (BD Biosci- ence) with random hexamer primers according to the manufacturer’s instructions. TOC1, LHY, CCA1,andGI transcript abundance was ACKNOWLEDGMENTS measured in each sample relative to UBQ10 using quantitative real- This research was initiated in A.J.M.’s group at the University of time PCR in a Rotor-Gene 3000 real-time PCR machine (Corbett Re- Warwick and pursued in A.H.’s group at the University of Liverpool. search). Each reaction contained 2 mL of cDNA product and 6 mLof A.H. and C.L. carried out the leaf movement analysis in Liverpool, A.H. in QuantiTect SYBR Green PCR mix (Qiagen) together with gene-specific Warwick. A.H. and P.D.G. carried out the luciferase analysis of the gi primers: UBQ10 forward primer, 59-CACACTCCACTTGGTCTTGCGT-39; mutants and cca1 and lhy mutants in Liverpool, A.H. and M.M.S. in UBQ10 reverse primer, 59-TGGTCTTTCCGGTGAGAGAGTCTT-39; TOC1 Warwick. Real-time PCR was performed by P.D.G. Modeling work was forward primer, 59-TCTTCGCAGAATCCCTGTGAT-39; TOC1 reverse carried out by J.C.W.L. The gi-611 and gi-596 mutants were isolated in a primer, 59-GCTGCACCTAGCTTCAAGCA-39; LHY forward primer, 59-ACG- screen performed by M.M.S., S.H., A.H., and S.J.D., and the flowering- AAACAGGTAAGTGGCGACA-39; LHY reverse primer, 59-TGGGAACAT- time measurement was carried out by J.P. We are grateful to Victoria CTTGAACCGCGTT-39; CCA1 forward primer, 59-GATGATGTTGAGG- Hibberd and Nazir Sharrif for expert technical assistance in Warwick, to CGGATG-39; CCA1 reverse primer, 59-TGGTGTTAACTGAGCTGTG- Ferenc Nagy and La´ szlo´ Kozma-Bogna´ r for sequencing the gi alleles, AAG-39; GI forward primer, 59-GGTCGACGGTTTCTCCAATCTA-39; and to N. Bueno del Carpio for reading the manuscript. M.M.S. GI reverse primer, 59-CGGACTATTCATTCCGTTCTTC-39. was supported by a PhD studentship from the Biotechnology and The UBQ10 and LHY primers have been described previously Biological Science Research Council (BBSRC), S.J.D. by a Department (Czechowski et al., 2004), as have the CCA1 and GI primers (Hall et al., of Energy Fellowship of the Life Sciences Research Foundation, and 2003) and the TOC1 primers (Farre et al., 2005). The efficiency value of S.H. in part by a postdoctoral fellowship from the Japan Society for the amplification for each set of primers was determined beforehand by Promotion of Science. Research at Liverpool was funded by BBSRC measuring the abundance of transcripts from a cDNA dilution series. Grant BBS/B/11125 and Royal Society Grant R4917/1 to A.H. Research Efficiency values were computed for each primer set using REST (http:// at Warwick was funded by BBSRC Awards G08667, G13967, and www.wzw.tum.de/gene-quantification/; Pfaffl et al., 2002). Each RNA G15231 to A.J.M. sample was assayed in triplicate, and RNAs were assayed from three biological repeats. The TOC1, LHY, CCA1,andGI transcript abundance levels were normalized to UBQ using Q-gene software (http://www. Received December 1, 2005; revised February 23, 2006; accepted March biotechniques.com/softlib/qgene.html; Muller et al., 2002). 7, 2006; published April 14, 2006.

Computational Methods

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